1. Field of the Invention
The present invention relates to a long-chain trans-prenyl diphosphate synthase gene, a plant transformed with an expression vector containing the gene, and a method for producing trans-1,4-polyisoprene using the plant.
2. Description of the Related Art
Polyisoprene (rubber), which is one of isoprenoid compounds, is classified into the cis-form and the trans-form according to the way in which isoprene units are polymerized. It is known that there are many plants that produce long-chain cis-polyisoprene (cis-1,4-polyisoprene), such as Hevea brasiliensis belonging to the family Euphorbiaceae, and Taraxacum and Lactuca indica belonging to the family Asteraceae. Among these, cis-1,4-polyisoprene produced by Hevea brasiliensis is commonly used commercially as a natural rubber (N. Ohya and T. Koyama, “Biosynthesis of natural rubber and other natural polyisoprenoids”, Biopolymers, (Germany), WILEY-VCH, 2001, vol. 2, p. 73-109). On the other hand, it is known that there are a small number of plants that naturally produce long-chain trans-polyisoprene (trans-1,4-polyisoprene), such as Eucommia ulmoides belonging to the family Eucommiaceae, Periploca sepium belonging to the family Apocynaceae, and Mimusops balata and Palaquium gutta belonging to the family Sapotaceae, but they are not commercially used (T. Bamba et al., “In-situ chemical analyses of trans-polyisoprene by histochemical staining and Fourier transform infrared microspectroscopy in a rubber-producing plant, Eucommia ulmoides Oliver”, Planta, 2002, vol. 215, p. 934-939). Among these, Eucommia ulmoides, which is a woody plant native to China, produces fibrous trans-1,4-polyisoprene. The leaves, bark, and peel of Eucommia ulmoides contain a large amount of trans-1,4-polyisoprene (T. Bamba et al.). However, currently, trans-1,4-polyisoprene is chemically synthesized, and used for outer layers of golf ball, plaster casts, sports protectors, and the like. Trans-1,4-polyisoprene is a thermoplastic elastomer having a low-melting point and high elasticity, and is also useful as an insulating material. Here, the word “natural rubber” commonly refers to a natural product-derived rubber in general, but may industrially refer to only a cis-rubber obtained from Hevea brasiliensis. It is not rare for higher plants to produce a rubber, and approximately 500 types of plants are confirmed to contain a rubber (“Regarding Rubber”, (online), Nihonkai Rubber Co., Ltd., (accessed on Sep. 2, 2008), the Internet <http://www.nihonkair.co.jp/semi02.htm>).
All natural isoprenoid compounds are biosynthesized using, as an intermediate, prenyl diphosphate in which isoprene units having five carbon atoms (C5) are continuously linked, and all prenyl diphosphates are biosynthesized by prenyl diphosphate synthases (K. Wang and S. Ohnuma, “Chain-length determination mechanism of isoprenyl diphosphate synthases and implications for molecular evolution”, TIBS, 1999, vol. 24, p. 445-4.51, and Tonetoshi Koyama and Kyozo Ogura, “Unlocking the mystery of natural rubber biosynthesis—Mechanism of construction of isoprene chains inside a living body”, Chemistry Today, 1990, vol. 237, p. 42-49). Prenyl diphosphate synthase is a general term for enzymes that produce prenyl diphosphate having a larger number of isoprene units (a longer chain length) than that of a primer substrate, by catalyzing a reaction that condenses isopentenyl diphosphate (IPP), which is a compound having five carbon atoms (C5), to prenyl diphosphate (an allyl substrate) functioning as a primer substrate (Japanese Laid-Open Patent Publication No. 2004-24275). The prenyl diphosphate synthase is also referred to as a prenyl transferase or a prenyl chain-elongating enzyme (Seiji Takahashi and Tanetoshi Koyama, “Molecular analysis of the enzymes participating in isoprenoid biosynthesis”, Kagaku To Seibutsu (Chemistry and Biology), 2005, vol. 43, p. 296-304).
IPP, which is a substrate for a prenyl diphosphate synthase, is biosynthesized by the mevalonate pathway or the like. Parts of the gene clusters for the enzymes participating in the mevalonate pathway have been clarified in various plants, such as Eucommia ulmoides. 
Prenyl diphosphate synthases can be classified into enzymes that catalyze a condensation reaction that forms an E form (trans-form) double bond, and enzymes that catalyze a condensation reaction that forms a Z form (cis-form) double bond, during condensation of IPP. Furthermore, prenyl diphosphate synthases may catalyze a reaction that further condenses IPP to prenyl diphosphate produced by a condensation reaction. The maximum length of isoprene chain that can be produced by such a condensation polymerization reaction of IPP (the maximum degree of IPP polymerization) is inherent to each prenyl diphosphate synthase. The hydrophobicity of a product changes depending on the isoprene chain length of the product, and, thus, the manner of expression of enzymatic activity significantly varies.
More specifically, prenyl diphosphate synthases can be classified into four types, a prenyl diphosphate synthase I (E-form short-chain prenyl diphosphate synthase), a prenyl diphosphate synthase II (E-form medium-chain prenyl diphosphate synthase), a prenyl diphosphate synthase III (E-form long-chain prenyl diphosphate synthase), and a prenyl diphosphate synthase IV (Z-form long-chain prenyl diphosphate synthase) (Japanese Laid-Open Patent Publication No. 2004-24275).
Examples of the prenyl diphosphate synthase I (E-form short-chain prenyl diphosphate synthase) include a geranyl diphosphate (GPP) synthase (C5→C10), a farnesyl diphosphate (FPP) synthase (C10→C15), and a geranyl geranyl diphosphate (GGPP) synthase (C15→C20). Here, for example, “C5→C10” refers to catalyzing a reaction that produces prenyl diphosphate having ten carbon atoms (C10) by condensing IPP having five carbon atoms to prenyl diphosphate functioning as a primer substrate having five carbon atoms (C5).
Examples of the prenyl diphosphate synthase II (E-form medium-chain prenyl diphosphate synthase) include a hexaprenyl diphosphate (HexPP) synthase (C15→C30) and a heptaprenyl diphosphate (HepPP) synthase (C16→C35).
Examples of the prenyl diphosphate synthase III (E-form long-chain prenyl diphosphate synthase) include an octaprenyl diphosphate (OctPP) synthase (C15→C40), a nonaprenyl diphosphate (NonPP) synthase (C10→C45), and a decaprenyl diphosphate (DecPP) synthase (C15→C50).
Examples of the prenyl diphosphate synthase IV (Z-form long-chain prenyl diphosphate synthase) include a Z-nonaprenyl diphosphate synthase (C15→C45), an undecaprenyl diphosphate (UPP) synthase (C15→C55), and a dehydrodolichyl diphosphate (deDolPP) synthase (C15→C85 to 105).
A rubber transferase gene (HRT2) is isolated from Hevea brasiliensis, which is a plant that produces a cis-rubber, and a protein that is encoded by the HRT2 gene is confirmed to have a cis-prenyl diphosphate-synthesizing activity that condenses IPP to rubber particles. Furthermore, the HRT2 gene is confirmed to complement the functional deficiencies of the dehydrodolichyl diphosphate synthases of a budding yeast (K. Asawatreratanakul et al., “molecular cloning, expression and characterization of cDNA encoding cis-prenyltransferases from Hevea brasiliensis”, Eur. J. Biochem., 2003, vol. 270, p. 4671-4680). However, it has not been reported that transformation of Hevea brasiliensis with an expression vector containing the HRT2 gene results in an increased content of cis-1,4-polyisoprene (cis-rubber) produced by Hevea brasiliensis. 
On the other hand, genes for long-chain trans-prenyl diphosphate synthases participating in the biosynthesis of a trans-rubber have not been isolated and identified from Eucommia ulmoides, Periploca sepium, Mimusops balata, and Palaquium gutta, which are plants that produce a trans-rubber. The inventors of the present invention isolated a prenyl transferase gene from Eucommia ulmoides (base sequence: GenBank Accession Number AB041626, and amino acid sequence: GenBank Accession Number BAB16687), but have not yet identified whether this gene encodes a cis-prenyl diphosphate synthase or a trans-prenyl diphosphate synthase, and whether this gene encodes a short-chain prenyl diphosphate synthase or a long-chain prenyl diphosphate synthase.
By the way, 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) is considered to be a key enzyme in the biosynthesis system of IPP functioning as a substrate for prenyl diphosphate synthases. When Arabidopsis thaliana is transformed with an expression vector containing DNA that encodes the catalyst domain of the HMGR (HMGR-CD), the transformed Arabidopsis thaliana has the total content of sterols that is approximately 3.6 times as large as that of the wild-type (D. Manzano et al., “The metabolic imbalance underlying lesion formation in Arabidopsis thaliana overexpressing farnesyl diphosphate synthase (isoform 1S) leads to oxidative stress and is triggered by the developmental decline of endogenous HMGR activity”, Planta, 2004, vol. 219, p. 982-992). Here, sterols are one of the isoprenoid compounds that are biosynthesized using IPP as a substrate.
For example, coenzyme Q10 is also known as one of the trans-form isoprenoid compounds. Wild-type Oryza sativa produces coenzyme Q9 using solanesyl diphosphate (in which nine isoprene units are polymerized) as an intermediate. When Oryza sativa is transformed with an expression vector containing DNA that encodes a Gluconobacter suboxydans-derived decaprenyl diphosphate (in which ten isoprene units are polymerized) synthase, the transformed Oryza sativa does not produce coenzyme Q9, but produces coenzyme Q10 using decaprenyl diphosphate as an intermediate (S. Takahashi et al., “Metabolic engineering of coenzyme Q by modification of isoprenoid side chain in plant”, FEBS Lett., 2006, vol. 580, p. 955-959). When Escherichia coli is transformed with an expression vector containing DNA that encodes a fungus-derived decaprenyl diphosphate synthase, the transformed Escherichia coli effectively produces coenzyme Q10 using decaprenyl diphosphate as an intermediate (International Publication Nos. 2002/092811 and 2002/040682).